Significance and Specific Aims: The organization of the photoreceptor is dependent on the proper biosynthesis, sorting, deposition and retention of its proteins in their functional domains. Recent advances in the molecular biological basis of several inherited retinal degenerations, including autosomal dominant retinitis pigmentosa arising from mutations of the rhodopsin and rds/peripherin genes demonstrate the importance of this concept in maintaining normal vision in man. Accordingly, we are exploring the process of sorting of rhodopsin from the Golgi apparatus to the outer segment. We have also studied the mechanism of cell death in inherited retinal dystrophies and discovered that it proceeds by a process resembling programmed cell death or apoptosis. Methods and Results: Frog retinal homogenates are fractionated to isolate post-Golgi membranes (PGM) in high purity that bear newly synthesized rhodopsin. The proteins on isolated PGM have been characterized. Several proteins of the visual transduction pathway including subunits of transducin and phosphodiesterase have been identified. In addition, by a combination of sequencing and immunochemical approaches, both alphaA2- and alphaB2-crystallins have been found to be synthesized by the retina and to be specifically associated with the rhodopsin-bearing PGM. This provides evidence for an extraordinary, new extralenticular function for these proteins in addition to their important role as chaperones in the lens where they assist in maintaining transparency. A set of small GTP binding rab proteins, structurally related to the ras oncogene have also been discovered including rab3, rab6, rab8 and at least two ARF proteins that can be ADP ribosylated. Research Plan: There are several additional members of this rab family still to be identified by their electrophoretic mobility, pI, and immunochemistry which should proceed readily since their structures are highly conserved and sequences of mammalian homologues are known. We now propose to evaluate the function of these rab proteins by a variety of approaches, primarily molecular, including the use of antisense constructs and blocking of "effector" domains by competition with synthetic prptides in reconstituted systems in vitro. The role of crystallins in transport of rhodopsin in photoreceptors will employ similar techniques as well as evaluating the impact of alteration of their phosphorylation state by phosphatase inhibitors, since only the unphosphorylated forms are membrane bound to PGM. Cell death in inherited retinal degenerations will be studied by a new technique of in situ end labeling of endonuclease cleaved DNA using TDT, biotinyl d-UTP and streptavidin conjugates. We seek additional long-term support to continue these studies of the fundamental basis of organization of photoreceptors and modest support to complete the initial studies of apoptosis as a fundamental new insight into the mechanism of cell death in retinal degenerations.